Alkylcellobiosides and arylcellobiosides have applications as chromogenic substrates or inhibitors of cellulases. Cellobioselipids, in turn, are high value products which are used as clean and biodegradable biosurfactants in detergent formulations. To date, these products are mainly synthesized via chemical or enzymatic glycosylation reactions.
Several chemical procedures have been described for the synthesis of glycosides. The most common procedures include the Fisher method and the Koenigs-Knorr method. However, the application of the Fisher method for synthesis of alkylbiosides is difficult because of concurrent alcoholysis of the interglycosidic linkage (Koto et al., 2004) and the Koenigs-Knorr method consists of a multi-step protocol that involves toxic intermediates and produces anomeric mixtures that need to be separated (Koto et al., 2004). Hence, the production of glycosides using classical chemical methods are laborious, have low yields and generate toxic waste.
Several publications describe the use of glycosylhydrolases (GHs) for the synthesis of glycosides (Basso et al., 2002; Gargouri et al., 2004). These enzymes are indeed interesting catalysts for enzymatic glycosylation as they have a relatively broad specificity. However, a major drawback is that they are not optimally suited for synthetic reactions, as their normal function is the breakdown (hydrolysis) of carbohydrates. Since the presence of water is necessary to maintain the enzymatic activity, but on the other hand causes substrate and product hydrolysis, the hydration level of the media has to be carefully controlled using solvents (Basso et al, 2002). As a result the obtained yields remain limited as glycosidases lose activity at low water activities.
Alternatively, glycosyltransferases can be used for glycosylation reactions. A major drawback, however, is that these enzymes require expensive nucleotide-activated carbohydrates (e.g. UDP-glucose) as glycosyldonor (Mendez & Salas, 2001; Fu et al., 2003). Consequently, this technology is likely to find application only for the targeted glycosylation of very high-value therapeutic proteins. Thus, the general industrial feasibility of using the latter enzymes for glycosylation reactions is very low. Hence, there is currently still a need to more efficiently produce glycosides such as alkylcellobiosides, arylcellobiosides and cellobioselipids at reduced cost and with a reduced environmental footprint.
Glycoside phosphorylases (GPs) catalyze the reversible breakdown of saccharide chains with the help of inorganic phosphate, resulting in a C1-phosphorylated monosaccharide and a saccharide of reduced chain length (Kitaoka et al., 2002). Because of the reversibility of this reaction, GPs can also be used for the synthesis of glycosidic bonds. In the synthetic direction, glucose-1-phosphate is used as donor and a hydroxylated compound as acceptor. The use of GPs for synthetic application has so far been explored by a limited number of groups. Most of them use GPs for the synthesis of rare carbohydrates. Kitao & Sekine (1992) have used sucrose phosphorylase with xylitol as the acceptor, resulting in the synthesis of glucosyl-xylitol. Aisaka et al. (2000) synthesised alpha-D-glucosyl-L-fucose using sucrose phosphorylase to transfer the glucosyl group from sucrose to L-fucose at position C-4. Okada et al. (2003) have recently described the synthesis of five novel oligosaccharides with kojibiose phosphorylase from Thermoanaerobacter brockii. 
Cellodextrin phosphorylases are GPs that catalyze the reversible phosphorolysis of cellooligosaccharides into α-glucose-1-phosphate (Glc1P) and cellodextrins with reduced chain length (Kitaoka et al., 1992 and Kitaoka et al., 2002). They are involved in the degradation of cellulosic biomass in vivo. Only two CDPs have so far been described: one from Clostridium stercorarium and one from Clostridium thermocellum. Both enzymes are only 22% identical at the protein level. For the enzyme from C. stercorarium, solely the phosphorolysis of cello-oligosaccharides has been reported (Reichenberger et al., 1997). The enzyme from C. thermocellum has been characterised more thoroughly (Sheth 1969, Arai et al., 1994, Samain et al., 1995, Kawaguchi et al., and Sheth & Alexander, 1998) but glycosylation reactions with alkyl/aryl glucosides or glucolipids have not been reported.
The present invention discloses the finding that CsCDP shows a surprisingly high acceptor specificity for alkyl beta-glucosides, alkyl beta-sophorosides, aryl beta-glucosides, aryl beta-sophorosides, glucolipids and sophorolipids. Hence, the latter enzyme is useful to synthesize for example alkylcellobiosides, arylcellobiosides, cellobiolipids, cellotriolipids, glucosophorolipids and cellobiosophorolipids while overcoming the problems related to classical chemical or enzymatic glycosylation reactions to produce said products. In addition, the latter enzyme can also be employed to produce corresponding lactosides such as lactolipids when α-galactose-1-phosphate instead (Gal1P) of α-glucose-1-phosphate is used as a donor.
Chromatograms of the various glycolipids produced by CDP. Product purification was performed after the reaction of Glc1P with glucolipid (A), of Glc1P with sophorolipid (B), and of Gal1P with glucolipid (C). The masses of the products obtained by LC/MS are also shown.